This application claims priority from British Application No. 9222156.3, filed Oct. 21, 1992 and British Application No. 9307028.2, filed Apr. 2, 1993.
The invention relates to the use of a class of thiol proteases as protective antigens against helminth parasites, namely the Cathepsin L-like proteases frequently released as excretory/secretory products by such parasites.
Each species of domestic animal can be parasitised by a number of different species of helminths, a process which usually causes disease. For example the parasitic trematode Fasciola hepatica is known to be the cause of the economically important disease fascioliasis in ruminants, such as cattle and sheep. The parasite enters the mammalian host by penetrating the gut wall and spends approximately seven weeks feeding on and burrowing through the liver mass before migrating into the bile duct. Following infection, development of immunity in the host is poor and resistance to reinfection in already infected hosts is only partial or non-existent. Other parasitic flukes include Fasciola gigantica and Dicrocoelium spp. and also Paramphistomum spp.
Problems are also caused by nematodes such as hookworms (e.g. Necator, Ancylostoma, Uncinaria and Bunostomum spp.).
Of the blood feeding nematodes the genus Haemonchus infects the lining of the abomasum of ruminants, causing anaemia and weight loss and if untreated frequently leads to death. Animals infected with the related non-blood feeding nematode Ostertagia similarly fail to thrive and may die if untreated.
Other parasitic worms of economic importance include the various species of the following helminth genera: Trichostrongylus, Nematodirus, Dictyocaulus, Cooperia, Ascaris, Dirofilaria, Trichuris and Strongylus. In addition to domestic livestock, pets and humans may also be infected, not infrequently with fatal results and helminth infections and infestations thus pose a problem of considerable worldwide significance.
Control of helminth parasites of grazing livestock currently relies primarily on the use of anthelmintic drugs combined with pasture management. Such techniques are often unsatisfactory firstly, because anthelmintic drugs may have to be administered frequently, secondly because resistance against anthelmintic drugs is becoming increasingly widespread and thirdly because appropriate pasture management is often not possible on some farms and even where it is, it can place constraints on the best use of available grazing.
Numerous attempts have been made to control helminth parasites of domestic animals by immunological means. With very few exceptions (e.g. the cattle lungworm, Dictyocaulus viviparus) this has not proved possible.
A vaccine against F.hepatica has been proposed in WO90/08819 comprising a glutathione-S-transferase from F. hepatica as antigenic material.
Bennett (UK Patent No. 2169606B) extracted various antigens from Fasciola organisms by a process which separates antigens specific to the juvenile stage from antigens present throughout the juvenile and adult stages.
It is known that in vitro cultured F. hepatica release protease enzymes which are capable of cleaving immunoglobulins with a papain or Cathepsin-B type of activity (Chapman and Mitchell, Vet. Parasitol. 11 (1982), p. 165-178). It has been suggested that these protease enzymes may assist in evading the immune response in combination with the known ability of the worms to slough off the surface glycocalyx thus shedding bound antibody (Hanna, Exp. Parasitol 50 (1980), p. 155-70). Furthermore crude in vitro excretory/secretory products can under some circumstances confer immunity on rats (Rajasekariah et al, Parasitol. 79 (1979), p. 393-400) perhaps by raising antibodies to such enzymes thus inhibiting them. However, the precise nature of the enzymes is far from clear.
A study of excretory/secretory proteases involving gelatin substrate polyacrylamide gel electrophoresis (GS-PAGE) (Dalton and Heffernan, Mol. Biochem. Parasitol. 35 (1989), p. 161-166) showed a number of cysteine proteases with a wide range of molecular weights and falling generally into two groups, namely from 27.5 KDa to 46 KDa active at pH 4.5xe2x80x38.0 and from 60 KDa to 88 KDa active at pH 3.0-4.5. It was suggested that the latter group might correspond to the immunoglobulin cleaving enzymes of Chapman and Mitchell and that autolysis and/or aggregation of one or more protease enzymes might be giving the multiple band structure.
Subsequently an HPLC procedure was used and three peaks resolved. Protein from the 15 kDa peak was found to have the ability to cleave IgG at an optimum of pH 4.5 (Heffernan et al, Biochem. Soc. Trans. 19 (1991), page 275).
Another study attempting to characterise the protease enzymes of adult F. hepatica is that of Rege et. al. (Mol. Biochem. Parasitol. 35 (1989), p. 89-96) in which a 14,500 Da protein was purified by cation exchange chromatography and molecular sieve HPLC. Maximal hydrolysis of the substrate CBZ-Phe-Arg-AFC was found at pH 6.0. Rege et al used lyophilised whole worms as the source of their protease so that it is not clear whether their protease is excreted or not. They speculated that the protease might be involved in immune evasion or nutrition.
A protease isolated from xe2x80x9cFasciola spp.xe2x80x9d has been reported by Yamasaki et al. (Japan J. Parasitol., 38 (1989), p. 373-384). The protease had a molecular weight of 27 kDa as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), was capable of hydrolysing haemoglobin and this hydrolysing activity was inhibited by cysteine protease inhibitors. Monoclonal antibodies specific for the protease could also inhibit the haemoglbin hydrolysis.
Other studies relating to proteases released by helminth parasites are Fagbemi and Hillyer, Vet. Parasitol. 40 (1991), p. 217-226 relating to Fasciola gigantica; Knox and Kennedy, Mol. Biochem. Parasitol. 28 (1988), p. 207-216 relating to Ascaris suum; and Yamakami and Hamajima, Comp. Biochem. Physiol. 87B (1987), p. 643-648 relating to Paragonimus westermani. 
It has now been found that the group of cysteine proteases disclosed by Dalton and Heffernan as having a range of molecular weights in the range 27.5-88 KDa on gelatin substrate (GS) PAGE can in fact be resolved as two proteases of 27 KDa and 29.5 by SDS-PAGE under reducing conditions; that these proteases are two distinct Cathepsin L-like activities as determined by substrate specificity, affinity for ion exchange columns and N-terminal sequencing; that the proteases also have the ability to cleave immunoglobulins; that immunisation of rabbits with purified proteases can stimulate antibodies capable of neutralizing the enzyme activity; and that this discovery opens up the possibility of an effective vaccine against helminth parasites and in particular F. hepatica using well-characterised purified protective antigens and avoiding the drawbacks in terms of toxicity and side-effects such as immune suppression or dominance which are inherent in the use of unresolved crude excretory/secretory products.
Accordingly a first aspect of the present invention provides a vaccine for use in combating a parasitic infestation of helminths in a mammal wherein the antigenic material comprises a protease having enzyme activity of the Cathepsin L type, in at least partially purified form, or an antigenic fragment or epitope thereof, together with a carrier and/or adjuvant.
The invention also provides a method of combating a parasitic infestation of helminths in a mammal comprising administering to said mammal a vaccine according to the invention as hereinbefore defined in an amount effective to combat said infestation.
The mammal is preferably a ruminant, for example cattle or sheep, but the vaccine and method of the invention may also find application in humans.
Preferably the Cathepsin L-like protease is derived from flukes such as Fasciola or Dicrocoelium, in particular from the liver fluke Fasciola hepatica. Alternatively it is preferred that the Cathepsin L-like protease should be capable of stimulating an immune response which will be effective against Fasciola or Dicrocoelium, in particular F. hepatica and F. gigantica, such Cathepsin L-like-molecules from other species as are capable of conferring a cross-protective immune response thus forming a particularly preferred aspect of the invention.
The F. hepatica Cathepsin L-like protease shown hereinafter to possess a molecular weight of approximately 27 KDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis under reducing conditions is particularly preferred for use in the vaccine and method of the invention. This protease also gives an apparent molecular weight by molecular sieve HPLC of 15 KDa. It will be referred to as Cathepsin L1.
The F. hepatica Cathepsin L-like protease shown hereinafter to possess a molecular weight of approximately 29.5 kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis under reducing conditions is also particularly preferred for use in the vaccine and as a novel protein itself forms a further aspect of the invention. This Cathepsin will be referred to as Cathepsin L2 where necessary to distinguish it from Cathepsin L1.
The Cathepsin L-like activity incorporated in the vaccine according to the invention is in at least partially purified form. Preferably the Cathepsin L-like activity comprises at least 75% of the total excretory/secretory proteins present in the vaccine and more preferably the Cathepsin L is at least 95% pure. It will be appreciated that once Cathepsin L of at least 95% purity has been obtained it can be admixed with one or more further purified antigenic proteins, including one or more further excretory/secretory proteins, to form a polyvalent vaccine.
Cathepsin L-like activity can be demonstrated by the ability to cleave the synthetic peptide substrate Z-phe-arg-AMC (benzyloxycarbonyl-L-phenylalanyl-L-arginyl-7-amido-4-methyl coumarin) combined with a relative inability to cleave the related peptides Z-arg-arg-AMC and Z-arg-AMC thus distinguishing the enzyme from Cathepsin B and Cathepsin H. Confirmation of the protease as a Cathepsin L can also be obtained by a comparison of the N-terminal amino acid sequence with the sequences of known Cathepsin L molecules.
Rat liver Cathepsin L, a mammalian Cathepsin L, has been shown to exist as a two chain protein (Ishidoh et al, FEBS Letters 223 (1987), pages 69-73). It is unclear whether or not a similar structure is present in non-mammalian Cathepsin L, although see the amino acid sequencing results presented hereinafter.
The vaccines according to the invention may be formulated with conventional carriers and/or adjuvants and the invention also provides a process for the preparation of the vaccines comprising bringing into association a purified protease having enzyme activity of the Cathepsin L type or an antigenic fragment or epitope thereof and one or more adjuvants or carriers. Suitable adjuvants include aluminium hydroxide, saponin (ISCOMs), muramyl dipeptide, mineral and vegetable oils, DEAE dextran, nonionic block copolymers or liposomes such as Novasomes (Trade Mark of Micro Vesicular Systems Inc.), in the presence of one or more pharmaceutically acceptable carriers or diluents. Carriers for peptide sequences corresponding to epitopes of Cathepsin L-like protease according to the invention can be proteins such as Hepatitis B core antigen multiple antigen peptide or lipopeptides such as tripalmitoyl-S-glycerylcysteinylserylserine (P3CSS). Suitable diluents include liquid media such as saline solution appropriate for use as vehicles. Additional components such as preservatives may be included.
Administration of the vaccine to the host species may be achieved by any of the conventional routes, e.g. orally or parenterally such as by intramuscular injection, optionally at intervals e.g. two injections at a 7-35 day interval. A suitable dose when administered by injection might be such as to give an amount of Cathepsin L-like protein within the range 10-500 xcexcg.
While the Cathepsin L-like protease for use in the vaccine according to the invention may be prepared by isolation from the excretory/secretory products of adult and/or juvenile helminths, it may also be convenient to prepare it by recombinant DNA techniques with the known advantages which such techniques give in terms of purity of product, scaling-up of production and reproducibility. Thus the invention also provides a Cathepsin L-like protease or a proenzyme therefor or an antigenic fragment or epitope thereof, produced by means of recombinant DNA techniques.
Additional aspects of the invention related to the above include DNA molecules encoding for Cathepsin L-like proteases or antigenic fragments or epitopes thereof; vectors containing one or more such DNA sequences; host cells, for example bacteria such as E. coli or more preferably eukaryotic cells, transformed by such vectors, for example by a baculovirus vector; and processes for preparing recombinant Cathepsin L-like protease or antigenic fragments or epitopes thereof comprising culturing such transformed host cells and isolating said Cathepsin L-like protease or fragment or epitope from the cultured cells. Since the tertiary structure of the Cathepsin L-like protease is important in the antibody response of a vaccinated animal eukaryotic expression systems are preferred as the tertiary structure will be more faithfully reproduced.
An alternative live or inactivated vaccine formulation may comprise an attenuated or virulent virus or a host cell, e.g. a microorganism such as a bacterium, having inserted therein a DNA molecule according to the invention for stimulation of an immune response directed against polypeptides encoded by the inserted nucleic acid molecule.
Additional antigenic materials may also be present in the vaccine thus giving an enhanced protective effect against the helminth parasite in question or a combined protective effect against one or more additional parasitic infestations.
A yet further aspect of the invention provides a monoclonal or polyclonal antibody capable of inducing immunity to a Cathepsin L-like protease in a mammal when administered to said mammal, the antibody having an affinity for the variable region of one or more further antibodies, said further antibodies having an affinity for said Cathepsin L.
This approach, the so-called xe2x80x9canti-idiotypexe2x80x9d approach, permits formulation of a vaccine which will dispense entirely with the original antigen and may offer even greater advantages in terms of safety, avoidance of side effects and convenience of manufacture.